Imperial College London/Notebook/10 September 2009

Testing

Diauxic growth 10th Sep

Protocol considerations

• Multiwell plate reader
  • Using script IGEM Abs, taking readings every 30 min

• Overnight culture cells were grown. (normal Top10)
• A volume of overnight cells were diluted into fresh M9 media

• 1ml of each secondary carbon source media (in M9) was prepared
• 200ul was added to 96 well plate as blank

• Write out well plate layout and do test runs of script *IMPORTANT*

• After about 5 hours, when cells were at a high enough OD (cloudy), cells were added 1:20 into the media
  • the cells and media were mixed well, and then 200ul was added into 96 well plates

• The plate reader was run overnight

Results

Download raw data

Conclusions

• Cells in Maltose grow slower, while the growth rate in the other secondary carbon sources are similar
• The cells appear to have continuous growth up till 7 hours. However, after that, the results become random. This is probably due to the evaporation problem

Suggestions/Improvements

• There is an initial spike. This should be a multiplate reader problem
  • Suggestion is to use a higher starting OD ~ 0.5 or 1

• The diauxic growth is using normal cells which grow on Strep. However, the growth profile might be significantly different on Amp (for our testing construct)
  • Therefore, use a cell with an Amp construct that is not expressed

• Try not to use any readings from the multiplate reader after around 8 hours

• To arrive at the switch point faster, 0.01% Glucose will be used instead for next experiment